592 Gut, 1992, 33, 592-596

Effect ofHelicobacterpylori infection on colloidal concentration in gastric mucus Gut: first published as 10.1136/gut.33.5.592 on 1 May 1992. Downloaded from

D J B Mufnoz, C Tasman-Jones, J Pybus

Abstract bacterpylori. Movement ofa drug into mucus will Necropsy gastric mucus infected with Helico- be determined by its intragastric availability. bacter pylon has a reduced capacity to con- Availability will depend upon various factors centrate colloidal bismuth subcitrate when including the drug's behaviour in solution. compared with non-infected mucus. Mucus It is not known whether non-infected and mounted in a modified in vitro diffusion Helicobacter pylon infected mucus differ in their chamber was bathed with colloidal bismuth capacity to contain colloidal bismuth subcitrate subcitrate solutions at different concentra- and little is known on the behaviour of colloidal tions and pH levels. Bismuth was measured by bismuth subcitrate in solution. atomic absorption spectrophotometry to In this in vitro study we have examined the assess intramucus colloidal bismuth subcitrate effect of Helicobacter pylon' infection on the concentrations. Bismuth concentrations in capacity ofgastric mucus to concentrate colloidal non-infected mucus were higher than in bismuth subcitrate and the behaviour ofcolloidal infected mucus at all bismuth subcitrate in acidic solutions. experimental colloidal bismuth subcitrate con- centrations and pH levels. Regardless of the infection status, the intramucus concentration Methods of colloidal bismuth subcitrate was dependent upon the concentration of the bathing solution SPECIMENS and independent of the pH and the mucus Mucus from 20 human stomachs was collected at thickness. Colloidal bismuth subcitrate solu- necropsy within 24 hours of death. All persons

bility in saline solution varied with pH. (18-75 years) had died ofacute trauma or sudden http://gut.bmj.com/ and was least soluble in the pH range 1 1 to unexpected medical collapse. Each stomach was 3-25 and more soluble above and below this pH opened along the greater curvature and food range. This study suggests that Helicobacter contents were removed by washing with normal pylon infection is associated with physico- saline. chemical changes in the gastric mucus with a Mucus was harvested by gently scraping the reduction in its capacity to concentrate col- gastric mucosa with a glass slide and stored at

loidal bismuth subcitrate. Such a reduction -20°C for later experiments. on September 23, 2021 by guest. Protected copyright. may compromise the attainment of optimum Immediately before harvesting, a gastric tissue colloidal bismuth subcitrate concentrations specimen was taken for Helicobacterpylori urease necessary for its bactericidal activity. detection by the camplobacter like organism test (CLO test, Delta West Limited, Western Aus- tralia).34 Gastric mucus plays an important role in A mucus sample was considered infected with mucosal protection.' Disruption in the Helicobacter pylori when the tissue specimen integrity of the blanket of mucus has been found showed positive urease activity within one hour in individuals with peptic ulcer.8"12 Helicobacter with the CLO test and non-infected when the pylori present in gastric mucus of the stomach tissue specimen showed no positive urease or duodenal gastric metaplasia is associated with activity within 24 hours. gastritis and peptic ulceration.'3 22 Helicobacter Department of pylori resides in gastric mucus but not within Medicine, School of epithelial cells.2"24 It may play a role in mucin COLLOIDAL BISMUTH SUBCITRATE Medicine, University of Auckland, Auckland, breakdown.25-27 CONCENTRATION IN GASTRIC MUCUS New Zealand Eradication of Helicobacter pylori by colloidal The modified in vitro diffusion chamber pre- D J B Munioz bismuth subcitrate is followed by histological viously used for ion exchange studies on mucus` C Tasman-Jones improvement of gastritis and healing of peptic was used to expose gastric mucus to colloidal Department of Clinical ulcer.242829 If infection recurs, ulcer relapse is bismuth subcitrate. Chemistry, Auckland frequent, particularly after one year.2430 31 Mucus (115 + 1 mg or 230 ± 1 mg, depending Hospital, Auckland, New A combination of colloidal bismuth subcitrate on mucus layer thickness) was held between two Zealand with one or two antibiotics - fQr fine gauze mesh discs Swiss J Pybus example, (Nybolt PA-22/17; is more Correspondence to: amoxycillin and/or , Silk Bolting Cloth Mfg Co Ltd, Zurich, Switzer- Dr C Tasman-Jones, successful than monotherapy in eradicating land) separated by a plastic ring to form a layer Department of Medicine, School of Medicine, Helicobacterpylor 24323 400 ,im or 800 iim thick. The mucus containing University of Auckland, Ulcer relapse after colloidal bismuth sub- cassette was held in the chamber by two rigid Private Bag, Auckland, New Zealand citrate and other antibiotics may be the result mesh discs (Tetex Mono PES 130/TWL/C; Accepted for publication of a failure of the drug(s) to reach sufficient Swiss Silk Bolting Cloth Mfg Co Ltd, Zurich, 5 August 1991 intramucus concentrations to eradicate Helico- Switzerland). One side of the mucus was bathed Effect ofHelicobacter pylori infection on colloidal bismuth subcitrate concentration in gastric mucus 593

with saline solution adjusted to pH 6.0 and the different pH's. For each pH, one 60 ml test tube other side with filtered solutions (Filter Paper 1, containing 45 ml saline and one whole commer- Whatman International Ltd, Maidstone, cial tablet of colloidal bismuth subcitrate was

England) of colloidal bismuth subcitrate (De- 'tumbled' in the Chiltern rotatory mixer (Chiltern Gut: first published as 10.1136/gut.33.5.592 on 1 May 1992. Downloaded from Nol®, Gist-Brocades, Delft, The Netherlands) Scientific, Germany) at a rate of 32 'tumbles' per at different concentrations (as bismuth) and minute. Disintegration was assessed visually and pH's as follows: halfand total disintegration times were recorded. Series 1: Colloidal bismuth subcitrate 93 To determine if the outer coating of the com- mg/l, pH 6.0, mucus layer 800 [im thick. mercial tablet affects disintegration, tablets were Series 2: Colloidal bismuth subcitrate 232 stripped of their coating and exposed to saline mg/l, pH 6-0, mucus layer 400 ,um thick. solutions under the same conditions as above. Series 3: Colloidal bismuth subcitrate 64 mg/l, pH 6.0, mucus layer 400 iim thick. Series 4: Colloidal bismuth subcitrate 64 STATISTICAL ANALYSIS mg/l, pH 1 5, mucus layer 400 [tm thick. Results for colloidal bismuth subcitrate concen- Series 5: Colloidal bismuth subcitrate 127 tration experiments were expressed as mean mg/l, pH 1 5, mucus layer 400 [im thick. (SD). A general linear approach to the analysis of The solutions were continuously circulated for variance37 was used to determine the statistical 90 minutes at a rate of 4 5 ml/min, room significance of differences in bismuth concen- temperature 2 1C. tration between non-infected and Helicobacter After each experiment the excess of colloidal pylori infected mucus. Significant mean and bismuth subcitrate solution was washed off with interaction effects were further investigated saline. The mucus was taken out of the chamber using Tukey's procedure to analyse differences and dried at 1 10°C for four hours. within pH's and mucus thickness. Type III sums The bismuth content of the samples was of squares are presented because the design was measured by atomic absorption (Graphite unbalanced. p Values of less than 0.05 were Furnace Atomic Absorption Spectrophotometer considered significant. SpectrAA-20; Melbourne, Australia) to assess the intramucus colloidal bismuth subcitrate concentrations. Results were expressed as [tg Results bismuth/g mucus on the assumption that all mucus samples contain 95% water.36 The possi- COLLOIDAL BISMUTH SUBCITRATE bility that Helicobacter pylon infection may CONCENTRATION IN GASTRIC MUCUS

modify the water content has not been con- The Table shows mean (SD) and range bismuth http://gut.bmj.com/ sidered in this study. concentrations in non-infected and Helicobacter pylon infected gastric mucus. Bismuth concentrations were higher in non- COLLOIDAL BISMUTH SUBCITRATE SOLUBILITY infected than in Helicobacter pylon infected Normal saline solutions were adjusted to dif- mucus at all experimental colloidal bismuth ferent pH's (E512 Metrohm Herisau pH meter, subcitrate concentrations and pH levels (F(1,62)=

Switzerland). Commercial tablets of colloidal 52.86, p=00001). on September 23, 2021 by guest. Protected copyright. bismuth subcitrate stripped of their outer coat- Regardless of the infection status, bismuth ing were ground to a homogeneous powder. concentrations varied in proportion to the con- Weighed samples of the powder were added to centration of the bathing solution and were no saline to give mixtures with the same weight/ different at mucus thickness 400 iim or 800 ,tm volume ratio. After colloidal bismuth subcitrate and at pH 1.5 or 6.0 (F(s,62)=l106, p=0.0389). was dissolved in the saline solution, samples In separate experiments, using unfiltered were obtained from the supernatant and colloidal bismuth subcitrate solutions and no fine analysed for bismuth content by atomic absorp- gauze mesh we found that a whitish precipitate tion spectrophotometry. Colloidal bismuth sub- was deposited on the mucus surface after the citrate solubility was estimated by calculating the experiments, especially at low pH's; bismuth difference between expected bismuth level and concentrations were up to five fold greater than bismuth in solution after sample preparation. bismuth levels obtained after using filtered In separate experiments we observed the dis- colloidal bismuth subcitrate solutions. integration ofcommercial colloidal bismuth sub- citrate tablets. Saline solutions were adjusted to COLLOIDAL BISMUTH SUBCITRATE SOLUBILITY Figure 1 shows percentages of colloidal bismuth subcitrate solubility estimated from bismuth TABLE Bismuth concentrations in non-infected and Helicobacter pylori infected gastric concentrations in supernatant samples. Solu- mucus bility in solution was >81% at pH 0.5 to 1-05; at 1 1 to and >88% at to CBS solution Non-infected HP-infected <50% pH 3-25 pH 3-75 Mucus 6-25. Minimum solubility (<15%) occurred in mgll (Bi) pH thickness Mean (SD) Range (n) Mean (SD) Range (n) the pH range 1 1 to 2-1. Two points of rapid 93 6-0 800* 74.2t (23-9) 33-116 (10) 37-2t (21-7) 9-72 (10) change from high precipitation to high solubility 232 6-0 400 147-9 (25-1) 121-186 (5) 88-2 (38-7) 33-130 (5) can be one between and 64 6-0 400 714 (29-2) 38-108 (5) 27-0 (12-1) 17-46 (5) described, pH 3-25 3-75 64 1-5 400 50-6 (12-1) 35-66 (5) 23-2 (6-6) 14-32 (5) and the other between pH 1.05 and 1 1. 127 1-5 400 95-7 (22-5) 64-121 (6) 65-2 (16-5) 33-78 (6) Half disintegration times for commercial are HP-infected=Helicobacter pylon infected; CBS=colloidal bismuth subcitrate; *=[Am; colloidal bismuth subcitrate tablets shown in t-= tg bismuth/g mucus, assuming that all mucus samples contain 951% water. Figure 2. At pH 0 8, 1 0 and 1 *1, coated colloidal 594 Muinoz, Tasman-Jones, Pybus

100 precipitates at pH <5. Lee4' found 40 to 50% o 6.25 colloidal bismuth subcitrate retained in solution 80 in the pH range 1[0 to 3 0. Our results are

similar, with <50% colloidal bismuth subcitrate Gut: first published as 10.1136/gut.33.5.592 on 1 May 1992. Downloaded from maintained in solution in the pH range 1 1 to 3 25. Minimum solubility (<15%) occurred in ._4 the range 1 1 to 2 1. The differences may be the 40 result of variations in technique. We prepared -0:3 40 different solutions for each pH level using com- U) mercial tablets while Lee titrated a sole sample 20 of a 'buffered solution of colloidal bismuth sub- citrate' to the different pH's. Colloidal bismuth subcitrate dissolves and/or precipitates when exposed to an aqueous solu- 0 1 2 3 4 5 6 7 tion. In keeping with the known effects ofpH on pH the sol gel transitions of colloids,42 we speculate Figure 1: Colloidal bismuth subcitrate solub ility in saline that at pH 6.0 the bismuth subcitrate colloid solution (in %) v pH. Solubility was >81% 4atpH 05to 105; dissolves and remains in solution as negatively <50% at pH 1 1 to 3.25 and >88% at pH 3.75 to 6.25. charged molecular complexes. When the pH is reduced to 1-5, most ofthe colloid would precipi- bismuth subcitrate tablets did noit disintegrate tate, but some bismuthoxy cations and bismuth even after two hours of constan 't'tumbling'; ions as free Bi3+ will be formed and remain in disintegration times were shorter fc r tablets from solution, this will be the case of the bathing which the coating had been strippi ed. There was solution in our experiments at pH 1.5 as the a significant change in disintel gration times precipitated colloidal bismuth subcitrate was between pH 1 1 and 1[2. removed by filtration. The ability of polyanions to selectively accumulate hydrogen ions into a zone of water Discussion immediately adjacent to the fixed charges43 offers Necropsy gastric mucus has the caipacity to take a possible explanation for the in vitro concentra- tion of colloidal bismuth subcitrate in mucus at up colloidal bismuth subcitrate dep)ending on the concentration of colloidal bismuthi subcitrate in pH 6.0. The bismuth subcitrate colloid in con- the bathing solution. This carpacity is not tact with mucus may concentrate intramucus affected by changes in bathing soluition pH if the hydrogen ions decreasing the pH of its micro- http://gut.bmj.com/ to a concentration is maintained conistant, but is environment level where it is precipitated to a source reduced when the mucus is iinfected with become for Bi3+ and bismuthoxy Helicobacter pylori. formation and accumulation in mucus. In The Campylobacter like organisnns test was the Helicobacter pylon' infection, ammonia decreases sole test used to diagnose Helicwobacter pylori the availability of hydrogen ions in mucus,44 infection. This test has been foundl to be reliable thus, reducing the accumulation of bismuth and rapid, with at three hours simiilar sensitivity cations. on September 23, 2021 by guest. Protected copyright. to Gram staining and with no falIse positives.3 The in vitro concentration of colloidal bis- A postmortem study found that the urease test muth subcitrate in mucus at pH 1 5 may depend is equally reliable to detect Heli cobacter pylot on the interaction ofbismuthoxy cations and Bi3+ presence when compared with oth(er tests.3 with the negatively charged residues ofsialic acid There is a paucity of literatui e on the be and ester sulphates present in the glycoprotein haviour of colloidal bismuth subcitrate in matrix.45 Gastric mucus contaminated with solution. Wieriks et al4l report that colloidal microorganisms other than Helicobacter pylon' bismuth subcitrate is highly solublLe in water and shows a diminished number of sialic acid resi- dues.- Helicobacter pylori induces changes in the physicochemical integrity of mucus25-27 and may >120 O0 similarly decrease these residues accounting for O Coated tab lets the reduced capacity of Helicobacter pylori * Uncoated ltablets infected mucus to concentrate colloidal bismuth 90 subcitrate when compared with non-infected -E E mucus at pH 1[5. Another explanation for the differences in 0 60 intramucus bismuth concentrations is an alteration ofcomposition and structure ofmucus caused by continued glycoprotein and lipid _4 digestion by Helicobacter pylon enzymes25-27 0) 30 between death, harvesting ofmucus samples and 0* the experiments. 15 00 0 We postulate that the precipitated and soluble 0@0 0 ,\ 9 forms of colloidal bismuth subcitrate have dif- 1 1 0 1 2 3 4 6 7 ferent therapeutic actions. Gastric ulcer healing pH is related to the formation of a protective layer of Figure 2: Halfdisintegration times ofcoatec1(0) and precipitated colloidal bismuth subcitrate bonded uncoated (0) commercial tablets ofcolloidaiI bismuth to serous secretions on the ulcer crater.4' 47-4 An subcitrate. Points represent the mean offour experiments. acid pH of the lumen should aid precipitation of Effect ofHelicobacter pylori infection on colloidal bismuth subcitrate concentration in gastric mucus 595

the layer. Helicobacter pylori associated gastritis capacity to concentrate colloidal bismuth sub- and duodenal ulcer healing depends on the citrate and that this capacity is reduced when capacity of mucus (mucus of gastric metaplasia Helicobacter pylorn are present. In vivo,

in the case of duodenal ulcer) to concentrate the reduced capacity of mucus to concentrate Gut: first published as 10.1136/gut.33.5.592 on 1 May 1992. Downloaded from bismuth cations either in solution or mobilised colloidal bismuth subcitrate may compromise from bismuth subcitrate colloid in contact with the attainment of optimal intramucus colloidal mucus (depending on luminal pH). bismuth subcitrate concentrations necessary The concentrations of bismuth in infected for its bactericidal activity. The behaviour of mucus at pH 1 5 (14 to 32 [tg/g; 16 to 36 Ftg/g colloidal bismuth subcitrate in acidic solutions calculated as Bi2O3) observed in the present suggests that the intragastric availability of the study will not be representative of the in vivo drug in solution may be low, further restricting situation because they were obtained after 90 satisfactory antibacterial intramucus concentra- minutes of bathing with solutions at a constant tions. concentration. In vivo, a fasting intragastric pH ofabout 1 5 ,"" gastric emptying of40% or more This study was supported by a grant from the Research Division, for liquid meals after 20 minutes,5455 the diluting Gist Brocades, The Netherlands. The authors would like to thank effects offood and the long disintegration time of Professor Philippa Wiggins of the Department of Medicine, University of Auckland Medical School, New Zealand, for her commercial tablets at pH <1 1 (Fig 2) will helpful discussions and Mr Greg Gamble of the Department restrict the intragastric availability of colloidal of Medicine, Auckland Hospital, New Zealand, for statistical bismuth subcitrate in solution. analysis. Intramucus bismuth concentrations falling below the mean inhibitory concentration of 1 Pfeiffer CJ. Experimental analytsis of hydrogen ion diffusion in gastrointestinal mucus glvcoprotein. Am 7 Phtsiol 1981; colloidal bismuth subcitrate for Helicobacter 240: G176-82. pylon' (4 to 32 ig/ml in vitro,24 5159) would 2 Slomiany BL, Piasek A, Sarosiek J, Slomianv A. The role of surface and intracellular mucus in gastric mucosal protection explain the failure ofcolloidal bismuth subcitrate against hydrogen ion. Scand 7 Gastroenterol 1985; 20: to always eliminate the organism. Bismuth con- 1191-6. 3 Somasundaram K, Ganguly AK. Gastric mucosal protection centrations in endoscopy biopsy samples after an during restraint stress in rats: alteration in gastric adherent oral dose of colloidal bismuth subcitrate606' will mucus and dissolved mucus in gastric secretion. Hepato- gastroenterology 1985; 32: 24-6. contain mucus-surface precipitated colloidal 4 Takeuchi K, Magee D, Critchlow J, Matthews J. Studies of bismuth subcitrate and will not represent intra- the pH gradient and thickness of frog gastric mucus. Gastroenterology 1983; 84: 331-40. mucus colloidal bismuth subcitrate. 5 Turner NC, Martin GP, Marriot C. The influence of natiVe The clinical efficacy of colloidal bismuth sub- porcine mucus gel on hydrogen ion diffusion: the effect of potentially ulcerogenic agents. ] Pharm Pharmnacol 1985; 37: citrate in healing peptic ulcers and gastritis is 776-80.

dependent on multiple mechanisms. Eradication 6 Williams SE, Turnberg LA. Demonstration of a pH gradient http://gut.bmj.com/ across mucus adherent to rabbit gastric mucosa: evidence for of Helicobacter pylori is an important factor. a 'mucus-bicarbonate' barrier. Gut 1981; 22: 94-6. Resolution of the gastritis when associated with 7 Williams SE, Turnberg LA. Retardation of acid diffusion by pig gastric mucus: a potential role in mucosal protection. peptic ulcer will play an important role in ulcer Gastroenterology 1980; 79: 299-304. healing, possibly through humoral mechanisms. 8 Batten JJ, Sidebotham RL, Spencer J, Baron JH. Changes to mucus glycoprotein structures in patients with gastritis Small intramucus colloidal bismuth subcitrate associated with gastric ulcer. Clin Sci 1989; 77: concentrations either in gastric and/or duodenal (suppl 21): 8P. 9 Sarosiek J, Piotrowski J, Gabrvelewicz A, Slomiany A, mucus may aid healing through stimulation of Slomiany BL. Alterations in mucin hvdrophobicitv and on September 23, 2021 by guest. Protected copyright. production,6266 enhancement in molecular form distribution with peptic ulcer. Gastro- enterologv 1989; 96: A441. the activity of epidermal growth factor,67-69 10 Sarosiek J, Piotrowski J, Gabrvelewicz A, Slomianv A, inhibition of the protease and lipase activities Slomiany BL. Changes in the macromolecular organization of gastric mucin with peptic ulcer: differences in phospho- of Helicobacter pylori towards gastric glyco- lipids and covalently bound fatty acids. Gastroenterology proteins,707' and possible detachment of micro- 1988; 94: A398. 11 Sarosiek J, Slomiany A, Gabryelewicz A, Slomianv BL. organisms from the gastric epithelium.24 Changes in the macromolecular organization and physical Preliminary results of a study conducted by properties of gastric mucus with peptic ulcer. [Abstract]. Gastroenterology 1987; 92: 1615. Salmon72 show increased early (four weeks) heal- 12 Younan F, Pearson J, Allen A, \'enables C. Changes in the ing of duodenal ulcers treated with a combi- structure of the mucous gel on the mucosal surface of the stomach in association with . Gastro- nation of colloidal bismuth subcitrate and enterology 1982; 82: 827-31. when compared with colloidal bis- 13 Andersen LP, Holk S, Povlsen CO, Elsborg L, Justesen T. Campylobacter pyloridis in peptic ulcer disease. I. Gastric muth subcitrate or cimetidine alone. Masoero and duodenal infection caused by C. pylon histopathologic et al5° observed the existence of some duodenal and microbiologic findings. Scand.7 Gastroenterol 1987; 22: 219-24. ulcer patients with intragastric pH values 14 Baverd(jrffer E, Oertel H, Lehn N, Kasper G, Mannes GA, steadily ranging around 10 throughout the 24 Sauerbruch T, Stolte M. Topographic association between active gastritis and Campvlobacter ptlri colonisation. .7 Chl hours, regardless of meals and of the time of the Pathol 1989; 42: 834-9. day. As Figure 1 shows, at both pH's (3 75 to 15 Buck GE, Gourlev WK, Lee WK, Subramanvam K, Latimer 1 JM, DiNuzzo AR. Relation of Campvy1obacter pvloridis to 6 25 and 0), colloidal bismuth subcitrate solu- gastritis and peptic ulcer. 7 Infec l)is 1986; 153: 664-9. bility will be favoured but, as speculated, the 16 Johnston BJ, Reed PI, Ali MH. Campvlobacter like organisms in duodenal and antral endoscopic biopsies: relationship to colloidal bismuth subcitrate forms in solution inflammation. G'2t !986; 27: 1132-7. will be different. Further studies will be neces- 17 Marshall BJ, Armstrong JA, McGechic DB, Glancy RJ. Attempt to fulfil Koch's postulates for pvloric antmlobahiter. sary to determine if the intragastric pH is Med 7 Anzsi 1985; 142: 436-9. important for a better success in eradicating 18 Marshall BJ, Warren JR. Unidientified curved bacilli in thc stomach of patients svith gastritis and peptic ulceration. Helicobacter pylori. l ancer 1984;i: 1311-5. The ecologic niche for Helicobacterpylori is the 19 MVorris A, Nicholson G. Ingestion of Camzpvlobacier pvloridis causes gastritis and raised fasting gastric pH. AmE .7 Cwastro- gastric mucus. For any antibacterial agent to eniero)l 1987; 82: 192-9. eradicate it, it must be able to penetrate the 20 Price AB, I.evi J, Dolbv J,Ml, D)unscombe PI., Smith A, Clark J, et al. C,ampvtlobacieXr pvloridis in peptic ulcer mucus layer in sufficient concentrations. We disease: microbiology, pathology, and scanning electron have shown that gastric mucus possesses the mtcroscopv. G,ui 1985; 26: 1183-8. 596 Munioz, Tasman-Jones, Pybus

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